左金方对幽门螺旋杆菌感染的人GES-1细胞增殖与凋亡的作用研究
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摘要
目的研究左金方对幽门螺旋杆菌引起人正常胃黏膜上皮(GES-1)细胞增殖与凋亡的影响。方法幽门螺旋杆菌以不同的感染复数(1∶1、50∶1、100∶l、200∶1、300∶1)感染GES-1细胞,于12、24、48 h后,CCK-8检测细胞的增殖活性;以不同质量浓度(0.5、1.0、2.0、4.0μg/m L)左金方作用幽门螺旋杆菌感染的GES-1细胞12、24、48 h后,收集细胞,CCK-8检测细胞的增殖活性,流式细胞仪和Western blotting检测细胞的凋亡率;Hochest染色检测凋亡细胞的形态学改变。结果幽门螺旋杆菌感染GES-1细胞后,感染复数越大,侵染时间越长,抑制细胞生长和促进细胞凋亡的作用越强,幽门螺旋杆菌以100∶1的感染复数作用人GES-1细胞12、24、48 h后,细胞存活率分别降低到(80.57±1.21)%、(70.04±3.21)%、(67.74±2.91)%,细胞的凋亡率分别增加到(23.74±1.71)%、(53.60±1.87)%、(70.67±2.87)%;与1.0μg/m L左金方共培养24 h后,细胞存活率上升到(97.67±1.04)%,细胞凋亡率降低到(31.04±1.02)%,与相应模型组对比,差异显著(P<0.01);Westernblotting结果表明,左金方各处理组细胞Caspase-3蛋白水平表达均明显降低;Hochest荧光染色,从形态学的角度进一步验证了这一结果。结论幽门螺旋杆菌感染人GES-1细胞后,可以抑制细胞增殖,诱导细胞凋亡,左金方可以降低幽门螺旋杆菌引起的细胞损伤,保护GES-1细胞。
Objective To study the effect of Zuojin Formula on proliferation and apoptosis of human normal gastric epithelial cells(GES-1) cells infected by Helicobacter pylori. Methods GES-1 cells were infected by H. pylori at different multiplicity of infection(1∶1, 50∶1, 100∶1, 200∶1, 300∶1) for 12, 24, 48 h; GES-1 cells infected by H. pylori were treated with different concentrations(0.5, 1.0, 2.0, 4.0 μg/mL) of Zuojin Formula, and the cells were harvested after 12, 24, and 48 h. The proliferation activity of cells was detected by CCK-8, and the apoptosis rate of GES-1 cells was measured by Anexin V-FITC apoptosis detecting kit and flow cytometry. Moreover, the expression of Caspase-3 was detected by Western blot and the morphological changes of apoptotic cells were detected by Hochest staining. Results After infected by H. pylori, the cell viability showed a descending trend while the apoptotic rate showed tendency to ascend, with the increasing of multiplicity of infection and infection time. GES-1 cells were treated with multiplicity of infection of H. pylori at 100∶1 for 12, 24, 48 h, and the cell viability decreased to(80.57 ± 1.21)%,(70.04 ± 3.21)%, and(67.74 ± 2.91)%, while the apoptotic rate increased to(23.74 ± 1.71)%,(53.60 ± 1.87)%, and(70.67 ± 2.87)%.After co-cultured with 1.0 μg/m L Zuojin Formula for 24 h, the cell viability increased to(97.67 ± 1.04)%, and the apoptotic rate decreased to(31.04 ± 1.02)%, and the difference was statistically significant compared with the corresponding model group(P < 0.01).The results of Western blotting showed that the protein expression of Caspase-3 of all treatment groups was obviously decreased. Based on the morphological point of view, this result was further verified. Conclusion GES-1 infected by H. pylori NCTC11637 can inhibit cell proliferation and induce apoptosis, while ZuoJinFang could protect GES-1 cells from cell damage.
Objective To study the effect of Zuojin Formula on proliferation and apoptosis of human normal gastric epithelial cells(GES-1) cells infected by Helicobacter pylori. Methods GES-1 cells were infected by H. pylori at different multiplicity of infection(1∶1, 50∶1, 100∶1, 200∶1, 300∶1) for 12, 24, 48 h; GES-1 cells infected by H. pylori were treated with different concentrations(0.5, 1.0, 2.0, 4.0 μg/mL) of Zuojin Formula, and the cells were harvested after 12, 24, and 48 h. The proliferation activity of cells was detected by CCK-8, and the apoptosis rate of GES-1 cells was measured by Anexin V-FITC apoptosis detecting kit and flow cytometry. Moreover, the expression of Caspase-3 was detected by Western blot and the morphological changes of apoptotic cells were detected by Hochest staining. Results After infected by H. pylori, the cell viability showed a descending trend while the apoptotic rate showed tendency to ascend, with the increasing of multiplicity of infection and infection time. GES-1 cells were treated with multiplicity of infection of H. pylori at 100∶1 for 12, 24, 48 h, and the cell viability decreased to(80.57 ± 1.21)%,(70.04 ± 3.21)%, and(67.74 ± 2.91)%, while the apoptotic rate increased to(23.74 ± 1.71)%,(53.60 ± 1.87)%, and(70.67 ± 2.87)%.After co-cultured with 1.0 μg/m L Zuojin Formula for 24 h, the cell viability increased to(97.67 ± 1.04)%, and the apoptotic rate decreased to(31.04 ± 1.02)%, and the difference was statistically significant compared with the corresponding model group(P < 0.01).The results of Western blotting showed that the protein expression of Caspase-3 of all treatment groups was obviously decreased. Based on the morphological point of view, this result was further verified. Conclusion GES-1 infected by H. pylori NCTC11637 can inhibit cell proliferation and induce apoptosis, while ZuoJinFang could protect GES-1 cells from cell damage.
引文
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[11]Agréus L,Hellstro?m P M,Talley N J,et al.Andreasson,towards a healthy stomach?Helicobacter pylori prevalence has dramatically decreased over 23 years in adults in a Swedish community[J].Unit Eur Gastroenterol J,2016,4(5):686-696.
[12]Vannarath S,Vilaichone R,Rasachak B,et al.Antibiotic resistant pattern of Helicobacter pylori infection based on molecular tests[J].Asian Pacific J Cancer Prevent,2016,17(1):285-287.
[13]徐方方,周洲.中药抗幽门螺旋杆菌感染研究现状[J].实用中医药杂志,2016,32(9):945-947.
[14]李江,成虹.不同中药提取物对幽门螺杆菌耐药菌株体外抗菌活性研究[J].现代中医临床,2015,22(2):21-23.
[15]陈奇.中成药名方药理与临床[M].北京:人民卫生出版社,1998.
[2]胡伏莲.幽门螺杆菌感染治疗中的问题及其处理策略[J].中国中西医结合杂志,2010,3(30):229-231.
[3]林凡松,王红,陶可胜,等.难治性幽门螺杆菌感染的中医研究进展[J].世界临床医学,2016,10(8):167-168.
[4]王献献,刘维明.中医药治疗幽门螺杆菌感染相关胃病研究进展[J].云南中医中药杂志,2016,37(9):84-85.
[5]钱平,杨秀伟.左金方的化学成分、抗肿瘤作用及体内过程研究进展[J].中国现代中药,2015,17(2):177-184.
[6]余惠旻,候梅轩,林敬斌,等.左金方体内外抑制幽门螺杆菌的实验研究[J].中国中医基础医学杂志,2017,23(3):405-407.
[7]方玲子,周红祖,余惠旻.左金方与反左金方抗MPP+诱导SH-SY5Y细胞凋亡作用的对比研究[J].中南药学,2015,13(7):708-712.
[8]周红祖,周禹练,余惠旻.左金方和5-FU对胃癌细胞生长及BAX/Bcl-2表达的调控研究[J].中国中医基础医学杂志,2016,22(6):777-791.
[9]邱菡,周红祖,周禹练,等.左金方联合5-FU抑制人胃癌细胞RF-48的增殖作用研究[J].中国中医基础医学杂志,2016,22(7):929-932.
[10]Yang J,Dai L X,Pan X,et al.Protection against Helicobacter pylori infection in BALB/c mice by oral administration of multi-epitope vaccine of CTB-UreI-UreB[J].Pathog Dis,2015,73(5):1-9.
[11]Agréus L,Hellstro?m P M,Talley N J,et al.Andreasson,towards a healthy stomach?Helicobacter pylori prevalence has dramatically decreased over 23 years in adults in a Swedish community[J].Unit Eur Gastroenterol J,2016,4(5):686-696.
[12]Vannarath S,Vilaichone R,Rasachak B,et al.Antibiotic resistant pattern of Helicobacter pylori infection based on molecular tests[J].Asian Pacific J Cancer Prevent,2016,17(1):285-287.
[13]徐方方,周洲.中药抗幽门螺旋杆菌感染研究现状[J].实用中医药杂志,2016,32(9):945-947.
[14]李江,成虹.不同中药提取物对幽门螺杆菌耐药菌株体外抗菌活性研究[J].现代中医临床,2015,22(2):21-23.
[15]陈奇.中成药名方药理与临床[M].北京:人民卫生出版社,1998.